Antibody inhibitors of gdf-8 and uses thereof

ABSTRACT

The disclosure provides novel antibodies against growth and differentiation factor-8 (GDF-8), including antibody fragments, which inhibit GDF-8 activity in vitro and in vivo. The disclosure also provides methods for diagnosing, preventing, or treating degenerative disorders of muscle, bone, or insulin metabolism.

This application claims priority to U.S. provisional patent applicationSer. No. 60/324,528, filed on Sep. 26, 2001.

FIELD OF THE INVENTION

This invention relates to inhibitors of Growth Differentiation Factor-8(GDF-8) proteins and methods of use for such inhibitors. Moreparticularly, the invention provides novel antibodies and antibodyfragments that are specifically reactive with GDF-8 proteins in vitroand in vivo. The invention is particularly useful for diagnosing,preventing, or treating human or animal disorders in which an increasein muscle tissue would be therapeutically beneficial. Exemplarydisorders include neuromuscular disorders (e.g., muscular dystrophy andmuscle atrophy), congestive obstructive pulmonary disease, musclewasting syndrome, sarcopenia, and cachexia; adipose tissue disorders(e.g., obesity); type 2 diabetes; and bone degenerative disease (e.g.,osteoporosis).

BACKGROUND OF THE INVENTION

Growth and Differentiation Factor-8 (GDF-8), also known as myostatin, isa member of the Transforming Growth Factor-beta (TGF-β) superfamily ofstructurally related growth factors, all of which possessphysiologically important growth-regulatory and morphogenetic properties(Kingsley et al. (1994) Genes Dev., 8: 133-46; Hoodless et al. (1998)Curr. Topics Microbiol. Immunol., 228: 235-72). GDF-8 is a negativeregulator of skeletal muscle mass, and there is considerable interest inidentifying factors which regulate its biological activity. For example,GDF-8 is highly expressed in the developing and adult skeletal muscle.The GDF-8 null mutation in transgenic mice is characterized by a markedhypertrophy and hyperplasia of the skeletal muscle (McPherron et al.(1997) Nature, 387: 83-90). Similar increases in skeletal muscle massare evident in naturally occurring mutations of GDF-8 in cattle (Ashmoreet al. (1974) Growth, 38: 501-507; Swatland and Kieffer (1994) J. Anim.Sci., 38: 752-757; McPherron and Lee (1997) Proc. Natl. Acad. Sci. USA,94: 12457-12461; and Kambadur et al. (1997) Genome Res., 7: 910-915).Since GDF-8 is expressed in both developing and adult muscles, it is notclear whether it regulates muscle mass during development or in adults.Thus, the question of whether or not GDF-8 regulates muscle mass inadults is important from a scientific and therapeutic perspective.Recent studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF-8 proteinexpression (Gonzalez-Cadavid et al. (1998) PNAS, 95: 14938-43). Inaddition, GDF-8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781).

A number of human and animal disorders are associated with loss orfunctional impairment of muscle tissue, including muscular dystrophy,muscle atrophy, congestive obstructive pulmonary disease, muscle wastingsyndrome, sarcopenia, and cachexia. To date, very few reliable oreffective therapies exist for these disorders. However, the terriblesymptoms associated with these disorders may be substantially reduced byemploying therapies that increase the amount of muscle tissue inpatients suffering from the disorders. While not curing the conditions,such therapies would significantly improve the quality of life for thesepatients and could ameliorate some of the effects of these diseases.Thus, there is a need in the art to identify new therapies that maycontribute to an overall increase in muscle tissue in patients sufferingfrom these disorders.

In addition to its growth-regulatory and morphogenetic properties inskeletal muscle, GDF-8 may also be involved in a number of otherphysiological processes, including glucose homeostasis in thedevelopment of type 2 diabetes and adipose tissue disorders, such asobesity. For example, GDF-8 modulates preadipocyte differentiation toadipocytes (Kim et al. (2001) BBRC, 281: 902-906).

There are also a number of conditions associated with a loss of bone,including osteoporosis, especially in the elderly and/or postmenopausalwomen. Currently available therapies for these conditions work byinhibiting bone resorption. A therapy that promotes new bone formationwould be a desirable alternative to or addition to, these therapies.

Like TGF-β-1, -2, and -3, the GDF-8 protein is synthesized as aprecursor protein consisting of an amino-terminal propeptide and acarboxy-terminal mature domain (McPherron and Lee, (1997) Proc. Natl.Acad. Sci. USA, 94: 12457-12461). Before cleavage, the precursor GDF-8protein forms a homodimer. The amino-terminal propeptide is then cleavedfrom the mature domain. The cleaved propeptide may remain noncovalentlybound to the mature domain dimer, inactivating its biological activity(Miyazono et al. (1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al.(1988) J. Biol. Chem., 263: 7646-7654; and Brown et al. (1990) GrowthFactors, 3: 35-43). It is believed that two GDF-8 propeptides bind tothe GDF-8 mature dimer (Thies et al. (2001) Growth Factors, 18:251-259). Due to this inactivating property, the propeptide is known asthe “latency-associated peptide” (LAP), and the complex of mature domainand propeptide is commonly referred to as the “small latent complex”(Gentry and Nash (1990) Biochemistry, 29: 6851-6857; Derynck et al.(1995) Nature, 316: 701-705; and Massague (1990) Ann. Rev. Cell Biol.,12: 597-641). Other proteins are also known to bind to GDF-8 orstructurally related proteins and inhibit their biological activity.Such inhibitory proteins include follistatin, and potentially,follistatin-related proteins (Gamer et al. (1999) Dev. Biol., 208:222-232). The mature domain is believed to be active as a homodimer whenthe propeptide is removed.

GDF-8 is highly conserved in sequence and in function across species.The amino acid sequence of murine and human GDF-8 is identical, as isthe pattern of mRNA expression (McPherron et al. (1997) Nature 387:83-90; Gonzalez-Cadavid et al. (1998) Proc. Natl. Acad. Sci. USA 95:14938-14943). This conservation of sequence and function suggests thatinhibition of GDF-8 in humans is likely to have a similar effect toinhibition of GDF-8 in mice.

GDF-8 is involved in the regulation of many critical biologicalprocesses. Due to its key function in these processes, GDF-8 may be adesirable target for therapeutic intervention. In particular,therapeutic agents that inhibit the activity of GDF-8 may be used totreat human or animal disorders in which an increase in muscle tissuewould be therapeutically beneficial, particularly muscle and adiposetissue disorders, bone degenerative diseases, neuromuscular disorders,and diabetes, as discussed above.

SUMMARY OF THE INVENTION

The present invention provides novel protein inhibitors comprisingantibodies and antibody fragments that are specifically reactive with amature GDF-8 protein, whether it is in a monomeric form, active dimericform, or complexed in the GDF-8 latent complex. In an embodiment of theinvention, the antibodies bind to an epitope on the mature GDF-8protein, which results in a reduction in one or more of the biologicalactivities associated with GDF-8, relative to a mature GDF-8 proteinthat is not bound by the same antibody. In an embodiment of theinvention, the presently disclosed antibodies reduce GDF-8 activityassociated with negative regulation of skeletal muscle mass and/or bonedensity.

The presently disclosed antibodies possess unique and unexpectedbiological properties. For instance, one of skill in the art wouldtypically expect good neutralizing antibodies to strongly bind to theactive GDF-8 protein in vitro, forming a stable inhibitory complex withthe protein. A neutralizing antibody also called an inhibitory antibody,having a high affinity for a particular protein will typically beexpected to provide higher levels of neutralization relative to a loweraffinity antibody to the same protein. However, quite unexpectedly, thepresent inventors have discovered antibodies that only weakly bind toand neutralize active GDF-8 protein in vitro, yet are effective in vivo.The discovery of such antibodies led, in turn, to the identification ofa specific site on GDF-8 to which the antibodies bind. It is thereforeexpected that any antibody specifically binding to the identified sitewould similarly possess in vivo neutralizing properties.

Additionally, the presently disclosed antibodies possess unique andunexpected properties. For example, the antibodies not only recognizemature GDF-8 protein in its monomeric and dimeric forms, but alsorecognize the intact GDF-8 latent complex.

The presently disclosed antibodies may be administered in atherapeutically effective dose to treat or prevent medical conditions inwhich an increase in muscle tissue mass or bone density would betherapeutically beneficial. Diseases and disorders that may be treatedby these GDF-8 antibodies include muscle or neuromuscular disorders suchas muscular dystrophy, muscle atrophy, congestive obstructive pulmonarydisease, muscle wasting syndrome, sarcopenia, and cachexia; adiposetissue disorders such as obesity; metabolic disorders such as type 2diabetes, impaired glucose tolerance, metabolic syndromes (e.g.,syndrome X), insulin resistance induced by trauma (e.g., burns); andbone degenerative disease such as osteoporosis, especially in theelderly and/or postmenopausal women. Additional metabolic bone diseasesand disorders amenable to treatment with these GDF-8 antibodies includelow bone mass due to chronic glucocorticoid therapy, premature gonadalfailure, androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.

In addition, the presently disclosed antibodies may be used as adiagnostic tool to quantitatively or qualitatively detect mature GDF-8protein or fragments thereof, regardless of whether it is in a monomericform, dimeric active form, or complexed in the GDF-8 latent complex. Forexample, the antibodies may be used to detect quantitatively orqualitatively mature GDF-8 protein in a cell, bodily fluid, tissue, oran organ. The presence or amount of mature GDF-8 protein detected isthen correlated with one or more of the medical conditions listed above.

The presently disclosed antibodies may be provided in a diagnostic kit.The kit may contain other components that aid the detection of matureGDF-8 protein, and help correlate the results with one or more of themedical conditions described above.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ Figure (if ID NO. applicable) Description 1 FIG. 16 JA-16 heavychain variable region AA sequence 2, 3, 5, 8 Antibody binding sites inprotein 4 DNA sequence for GDF-8 (accession no. xm_030768) 6 Nucleicacid sequence encoding SEQ ID NO: 1 7 DNA sequence for BMP-11 (accessionno. xm_049170) 9 B1 peptide, derived from BMP-11 10 G1 peptide, derivedfrom GDF-8 11-13, 65, 105, FIG. 1 Synthetic peptides derived from SEQ ID114, 129 NO: 14 14 A cysteine-to-serine mutated sequence of matureGDF-8. 15 FIG. 3 mature GDF-8 AA sequence 16 FIG. 3 BMP-11 AA sequence18 JA-16 eptitope region from GDF-8 17-64 FIG. 6A Overlapping 13-merpeptides corresponding to portions of the GDF-8 sequence 65 BiotinylatedN-terminal peptide derived from GDF-8 66-104, Mutated versions of SEQ IDNO: 18 106-113, 115-128 130 AA sequence of GDF-8 propeptide (accessionno. xp_030768) 131 AA sequence of BMP-11 propeptide (accession no.xp_049170)

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the GDF-8 synthetic peptides (SEQ ID NOS: 11-13, 65, 105,114, and 129, all derived from SEQ ID NO:14) used to characterize thebinding specificity of JA-16. The underlined amino acids indicatepositions where native cysteines have been replaced with serines.

FIG. 2 shows the binding of JA-16 to the GDF-8 synthetic peptides.

FIG. 3 indicates the differences in the amino acid sequences of matureGDF-8 (SEQ ID NO:15) and BMP-11 (SEQ ID NO:16).

FIG. 4 shows a comparison of the binding characteristics of JA-16 to G1peptide (SEQ ID NO:10, a peptide derived from GDF-8) conjugated tobovine serum albumin (BSA) and B1 peptide (SEQ ID NO:9, a peptidederived from BMP-11) conjugated to BSA.

FIG. 5 shows a comparison of the binding characteristics of JA-16 toG1-BSA (SEQ ID NO:10) after JA-16 is preincubated with G1, B1, GDF-8, orBMP-11.

FIGS. 6A and B show the mapping studies of JA-16 binding usingoverlapping 13-mer synthetic peptide sequences from GDF-8.

FIGS. 7A and B show the results of a deletion and substitution analysisof the JA-16 epitope region from GDF-8,Gly-Leu-Asp-Ser-Asp-Glu-His-Ser-Thr-Glu-Ser-Arg-Ser (SEQ ID NO:18),using spot synthesis.

FIG. 8 shows the binding of biotinylated GDF-8 to the ActRIIB receptor.

FIG. 9 shows the inhibition of biotinylated GDF-8 binding to the ActRIIBreceptor in the presence and absence of JA-16.

FIG. 10 shows a reporter gene assay assessing the neutralizing effect ofJA-16 on the activity of GDF-8 in vitro.

FIG. 11 shows the in vivo effect of JA-16 in mice during a 4 week study.Seven week-old female BALB/c mice were treated for four weeks with JA-16by intraperitoneal injection at 50 mg/kg twice weekly. The graph on theleft shows the change in lean and fat mass during the treatment periodas measured by dexascan (dual energy x-ray) analysis. The graph on theright shows the mass of dissected tissues. A statistically significantdifference, p<0.01 for a student test, is indicated by an asterisk.

FIG. 12 shows the in vivo effect of JA-16 on total body mass of miceduring a 14-week study. Male C57BL mice used in this study were eitherwild type at the agouti locus (a) or carried the lethal yellow mutation(Ay) at that locus. The Ay mutation causes adult onset obesity anddiabetes. Young adult mice were treated with weekly intraperitonealinjections of 60 mg/kg of JA-16 or control antibody. In addition, at thestart of the treatment period, mice were loaded with 60 mg/kgintraperitoneally and 10 mg/kg intravenously of the same antibody. Thesegraphs show the weekly body weight for each group of mice. The errorbars show the standard error of the mean for each data point.

FIG. 13 shows the in vivo effect of JA-16 on total muscle mass in miceduring a 14 week study. At the end of the study, muscles were dissectedand weighed. These graphs show the average muscle mass for each group ofmice. A statistically significant difference, p<0.01 for a student test,is indicated by an asterisk.

FIG. 14 shows the in vivo effect of JA-16 on total fat mass in miceduring a 14-week study. At the end of the study, fat pads were dissectedand weighed. These graphs show the average fat pad mass for each groupof mice.

FIG. 15 shows the in vivo effect of JA-16 on blood glucose levels inmice during a 14 week study. After 12 weeks of treatment, the C57BL-Ay/amice were fasted overnight and their blood glucose was measured.

FIG. 16 shows the amino acid sequence of the JA-16 heavy chain variableregion (SEQ ID NO:1). The complimentarity determining regions (CDRs) areunderlined. The corresponding nucleic acid sequence is provided in SEQID NO:6.

FIG. 17 shows an in vivo comparison of Myo-19 and JA-16. Seven week-oldfemale C57B6/scid mice were treated for five weeks with JA-16, Myo-19,or vehicle by intraperitoneal injection. At the end of the study,muscles were dissected and weighed. These graphs show the average musclemass for each group of mice. A statistically significant difference,p<0.01 for a student t-test, is indicated by an asterisk.

FIG. 18 shows results from the immunoprecipitation of GDF-8 with JA-16and Myo-19.

FIGS. 19A and 19B show results of JA-16 treatment in BALB/c female micefor eight weeks. Mice were 21 months or 4 months of age at the end ofthe study. (19A) Dissected quadriceps mass for JA-16 treated and vehicletreated mice at the end of the study. (19B) Forelimb strength determinedby a grip test for JA-16 treated and vehicle treated mice after sevenweeks of treatment. Each bar or data point indicates the average valuefor the indicated group±the standard error; (**) indicates that p<0.01for Student's t-test comparing the JA-16 group to the vehicle group; n=8for each group.

FIGS. 20A-20D show results of JA-16 treatment in mdx mice. (20A) JA-16treated mice had significantly increased EDL weight compared to mdxcontrols (19.72±0.50 vs. 14.63±0.69 mg; n=12; p<0.0001). (20B) JA-16treated mice had significantly increased muscle mass to body weightratio (EDL weight/body weight) as compared to control (0.6±0.02 vs.0.5±0.02; n=12; p<0.014). (20C) JA-16 treated mice generatedsignificantly greater force during isometric twitch contraction ascompared to control (177.32±8.37 vs. 132.38±12.45 mN; n=12; p<0.03).(20D) JA-16 treated mice generated significantly greater force duringisometric tetanic contraction compared to control (491.23±16.34 vs.370.74±19.21 mN; n=12; p<0.003).

DEFINITIONS

The term “antibody” refers to one or more polyclonal antibodies,monoclonal antibodies, antibody compositions, antibodies having mono- orpoly-specificity, humanized antibodies, single-chain antibodies,chimeric antibodies, CDR-grafted antibodies, antibody fragments such asFab, F(ab′)₂, Fv, and other antibody fragments which retain the antigenbinding function of the parent antibody.

The term “chimeric antibodies” refers to molecules in which a portion ofthe heavy and/or light chain is identical or homologous to correspondingsequences from a particular species (or belonging to a particularantibody class or subclass), while the remainder of the chain(s) isidentical or homologous to corresponding sequences derived from adifferent species (or belonging to a different antibody class orsubclass). Such chimeric antibodies are described by Morrison, et al.(1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855.

The term “epitope” refers to a molecule or portion of a molecule that iscapable of specifically reacting with an anti-GDF-8 monoclonal antibody.Epitopes may comprise proteins, protein fragments, peptides,carbohydrates, lipids, or other molecules, but are most commonlyproteins, short oligopeptides, or combinations thereof.

The terms “GDF polypeptide” and “GDF protein” refer generally to anygrowth and differentiation factors that are structurally or functionallyrelated to GDF-8.

The term “GDF inhibitor” includes any agent capable of inhibitingactivity, expression, processing, or secretion of a GDF protein. Suchinhibitors include proteins, antibodies, peptides, peptidomimetics,ribozymes, anti-sense oligonucleotides, double-stranded RNA, and othersmall molecules which specifically inhibit the GDF proteins.

The terms “GDF-8” or “GDF-8 protein” refer to a specific growth anddifferentiation factor. The terms include the full length unprocessedprecursor form of the protein, as well as the mature and propeptideforms resulting from post-translational cleavage. The terms also referto any fragments of GDF-8 that maintain the known biological activitiesassociated with the protein, as discussed herein, including sequencesthat have been modified with conservative or non-conservative changes tothe amino acid sequence.

These GDF-8 molecules may be derived from any source, natural orsynthetic. The human form of mature GDF-8 protein is provided in SEQ IDNO:15. However, the present invention also encompasses GDF-8 moleculesfrom all other sources, including GDF-8 from bovine, chicken, murine,rat, porcine, ovine, turkey, baboon, and fish. These various GDF-8molecules have been described in McPherron et al. (1997) Proc. Natl.Acad. Sci. USA, 94: 12457-12461.

“Mature GDF-8” refers to the protein that is cleaved from thecarboxy-terminal domain of the GDF-8 precursor protein. The mature GDF-8may be present as a monomer, homodimer, or in a GDF-8 latent complex.Depending on the in vivo or in vitro conditions, an equilibrium betweenany or all of these different forms may exist. GDF-8 is believed to bebiologically active as homodimer. In its biologically active form, themature GDF-8 is also referred to as “active GDF-8.”

“GDF-8 propeptide” refers to the polypeptide that is cleaved from theamino-terminal domain of the GDF-8 precursor protein. The GDF-8propeptide is capable of binding to the propeptide binding domain on themature GDF-8.

“GDF-8 latent complex” refers to the complex of proteins formed betweenthe mature GDF-8 homodimer and the GDF-8 propeptide. It is believed thattwo GDF-8 propeptides associate with a GDF-8 homodimer to form aninactive tetrameric complex. The latent complex may include other GDF-8inhibitors in place of or in addition to one or more of the GDF-8propeptides.

The phrase “GDF-8 inhibitor” includes any agent capable of inhibitingthe activity, expression, processing, or secretion of GDF-8 protein.Such inhibitors include proteins, antibodies, peptides, peptidomimetics,ribozymes, anti-sense oligonucleotides, double-stranded RNA, and othersmall molecules that specifically inhibit the activity of GDF-8 protein.Such inhibitors are said to “neutralize” or “reduce” the biologicalactivity of GDF-8 protein.

The phrase “GDF-8 activity” refers to one or more of growth-regulatoryor morphogenetic activities associated with active GDF-8 protein. Forexample, active GDF-8 is a negative regulator of skeletal muscle. ActiveGDF-8 can also modulate the production of muscle-specific enzymes (e.g.,creatine kinase), stimulate myoblast cell proliferation, and modulatepreadipocyte differentiation to adipocytes.

The terms “isolated” or “purified” refer to a molecule that issubstantially free of its natural environment. For instance, an isolatedprotein is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which it isderived. The phrase “substantially free of cellular material” refers topreparations where the isolated protein is at least 70% to 80% (w/w)pure, optionally at least 80%-89% (w/w) pure, optionally 90-95% pure;and optionally at least 96%, 97%, 98%, 99% or 100% (w/w) pure.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, sheep, pigs, cows, etc. Themammal is human in one embodiment of the invention.

The term “monoclonal antibody” refers to one or more antibodies from asubstantially homogeneous antibody population that is directed against asingle antigenic epitope. The term encompasses humanized antibodies,single-chain antibodies, chimeric antibodies, CDR-grafted antibodies,antibody fragments such as Fab, F(ab′)₂, Fv, and other antibodyfragments which retain the antigen binding function of the parentantibody.

Furthermore, the term “monoclonal antibody” is not limited to anyparticular species or source of the antibody, or the manner by which itis made. Monoclonal antibodies may be made via traditional hybridomatechniques (Kohler and Milstein (1975) Nature, 256: 495-499),recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage antibodylibraries (Clackson et al. (1991) Nature, 352: 624-628; Marks et al.(1991) J. Mol. Biol., 222: 581-597). Monoclonal antibodies of anymammalian or non-mammalian species can be used in this invention. Forexample, the antibodies may be derived from primates (e.g., human,orangutan, etc.), avian (e.g., chicken, turkey, etc.), bovine, murine,rat, porcine, ovine, or fish. In one embodiment of the invention, theantibodies are of rat, murine, or human origin.

The terms “neutralize” and “neutralizing” refer to a reduction in theactivity of GDF-8 by a GDF-8 inhibitor, relative to the activity of aGDF-8 molecule that is not bound by the same inhibitor. Thus, a“neutralizing” antibody reduces the activity of GDF-8 relative to theactivity of a GDF-8 molecule not bound by the same antibody. Theactivity of the GDF-8 protein, when bound by one or more of thepresently disclosed GDF-8 inhibitors (e.g., the presently disclosedantibodies), is reduced at least about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, or 55%, optionally at least about 60%, 62%, 64%, 66%,68%, 70%, 72%, 72%, 76%, 78%, 80%, 82%, 84%, 86%, or 88%, optionally atleast about 90%, 91%, 92%, 93%, or 94%, and optionally at least 95% to100% relative to a GDF-8 protein that is not bound by one or more of thepresently disclosed GDF-8 inhibitors.

The term “specific interaction,” or “specifically binds,” or the like,means that two molecules form a complex that is relatively stable underphysiologic conditions. The term is also applicable where, e.g., anantigen-binding domain is specific for a particular epitope, which iscarried by a number of antigens, in which case the specific bindingmember carrying the antigen-binding domain will be able to bind to thevarious antigens carrying the epitope. Specific binding is characterizedby a high affinity and a low to moderate capacity. Nonspecific bindingusually has a low affinity with a moderate to high capacity. Typically,the binding is considered specific when the affinity constant K_(a) ishigher than 10⁶M⁻¹, or can be higher than 10⁸M⁻¹. If necessary,non-specific binding can be reduced without substantially affectingspecific binding by varying the binding conditions. Such conditions areknown in the art, and a skilled artisan using routine techniques canselect appropriate conditions. The conditions are usually defined interms of concentration of antibodies, ionic strength of the solution,temperature, time allowed for binding, concentration of non-relatedmolecules (e.g., serum albumin, milk casein), etc. Exemplary conditionsare set forth in Example 4.

The term “TGF-β superfamily” refers to a family of structurally -relatedgrowth factors, all of which are endowed with physiologically importantgrowth-regulatory and morphogenetic properties. This family of relatedgrowth factors is well known in the art (Kingsley et al. (1994) GenesDev., 8: 133-146; Hoodless et al. (1998) Curr. Topics Microbiol.Immunol., 228: 235-272). The TGF-β superfamily includes BoneMorphogenetic Proteins (BMPs), Activins, Inhibins, Mullerian InhibitingSubstance, Glial-Derived Neurotrophic Factor, and a still growing numberof Growth and Differentiation Factors (GDFs), such as GDF-8 (myostatin).Many of these proteins are related in structure to GDF-8, such asBMP-11; and/or activity, such as activin.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatment mayinclude individuals already having a particular medical disorder as wellas those who may ultimately acquire the disorder (i.e., those needingpreventative measures).

DETAILED DESCRIPTION OF THE INVENTION Antibodies

Intact antibodies are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains (Clothia et al. (1985) J. Mol. Biol., 186:651-663; Novotny and Haber (1985) Proc. Natl. Acad. Sci. USA, 82:4592-4596).

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgA1 and IgA2 for IgA; IgGI, IgG2, IgG3, IgG4 for IgGin humans, and IgG1, IgG2a, IgG2b, and IgG3 for IgG in mouse. Theheavy-chain constant domains that correspond to the major classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known in the art.

For a review of the antibody structure, see Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.Briefly, each light chain is composed of an N-terminal variable (V)domain (V_(L)) and a constant (C) domain (C_(L)). Each heavy chain iscomposed of an N-terminal V domain, three or four C domains, and a hingeregion. The C_(H) domain most proximal to V_(H) is designated as C_(H)1.The V_(H) and V_(L) domain consist of four regions of relativelyconserved sequence called framework regions (FR1, FR2, FR3, and FR4),which form a scaffold for three regions of hypervariable sequence(complementarity determining regions, CDRs). The CDRs contain most ofthe residues responsible for specific interactions with the antigen.CDRs are referred to as CDR1, CDR2, and CDR3. Accordingly, CDRconstituents on the on the heavy chain are referred to as H1, H2, andH3, while CDR constituents on the light chain are referred to as L1, L2,and L3. CDR3 is the greatest source of molecular diversity within theantibody-binding site. H3, for example, can be as short as two aminoacid residues or greater than 26. The locations of immunoglobulinvariable domains in a given antibody may be determined as described, forexample, in Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, eds. Kabat et al., 1991.

Antibody diversity is created by the use of multiple germline genesencoding variable regions and a variety of somatic events. The somaticevents include recombination of variable gene segments with diversity(D) and joining (J) gene segments to make a complete VH region and therecombination of variable and joining gene segments to make a completeVI. region. The recombination process itself is imprecise, resulting inthe loss or addition of amino acids at the V(D)J junctions. Thesemechanisms of diversity occur in the developing B cell prior to antigenexposure. After antigenic stimulation, the expressed antibody genes in Bcells undergo somatic mutation. Based on the estimated number ofgermline gene segments, the random recombination of these segments, andrandom V_(H)-V_(L) pairing, up to 1.6×10⁷ different antibodies could beproduced (Fundamental Immunology, 3rd ed., ed. Paul, Raven Press, NewYork, N.Y., 1993). When other processes which contribute to antibodydiversity (such as somatic mutation) are taken into account, it isthought that upwards of 1×10¹⁰ different antibodies could be generated(Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, SanDiego, Calif., 1995). Because of the many processes involved ingenerating antibody diversity, it is unlikely that independently derivedmonoclonal antibodies with the same antigen specificity will haveidentical amino acid sequences.

Antibodies may be raised against any portion of a protein which providesan antigenic epitope. In one embodiment of the invention, the presentlydisclosed antibodies specifically bind to an epitope on a proteinbelonging to the superfamily of TGF-β proteins. The protein isoptionally a Bone Morphogenetic Proteins (BMP), Activin, Inhibin,Mullerian Inhibiting Substance, Glial-Derived Neurotrophic Factor, orGrowth and Differentiation Factors (GDFs). Optionally, the protein isBMP-11, Activin, or GDF-8. The protein is optionally a mature GDF-8protein.

In an embodiment, the presently disclosed antibodies bind to a matureGDF-8 protein as set forth in SEQ ID NO:15; optionally between aminoacid 1 and amino acid 50; optionally between amino acid 1 and amino acid25; and optionally between amino acid 1 and 15 of SEQ ID NO:15.

In another embodiment, the presently disclosed antibodies specificallybind to the sequence Asp-Glu-His-Xaa-Thr (SEQ ID NO:2) in any one of theproteins belonging to the TGF-β superfamily, where Xaa is Ala, Gly, His,Met, Asn, Arg, Ser, Thr, or Trp. Optionally, the antibodies specificallybind to the peptide sequence Asp-Glu-His-Xaa-Thr (SEQ ID NO:2) in GDF-8,where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, or Trp. Optionally,the antibodies specifically bind to Asp-Glu-His-Ser-Thr (SEQ ID NO:3) inthe mature GDF-8 protein (SEQ ID NO:15).

Optionally, the presently disclosed antibodies specifically bind to thepeptide sequenceAsp-Phe-Gly-Leu-Asp-Cys-Asp-Glu-His-Xaa-Thr-Glu-Ser-Arg-Cys (SEQ IDNO:5) in any one of the proteins belonging to the TGF-β superfamily,where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, or Trp. Optionally,the antibodies specifically bind to the peptide sequenceAsp-Phe-Gly-Leu-Asp-Cys-Asp-Glu-His-Xaa-Thr-Glu-Ser-Arg-Cys (SEQ IDNO:5) in GDF-8, where Xaa is Ala, Gly, His, Met, Asn, Arg, Ser, Thr, orTrp. Optionally, the antibodies specifically bind to the peptidesequence Asp-Phe-Gly-Leu-Asp-Cys-Asp-Glu-His-Ser-Thr-Glu-Ser-Arg-Cys(SEQ ID NO:8) in the mature GDF-8 protein (SEQ ID NO:15).

The GDF-8 protein to which the presently disclosed antibodies mayspecifically bind is optionally at least about 75%-80% identical to SEQID NO:15, optionally at least about 81% to about 85% identical to SEQ IDNO:15, optionally at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,or 94% identical to SEQ ID NO:15, and optionally at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15. The GDF-8 proteinoptionally comprises SEQ ID NO:15.

In an alternative embodiment, the presently disclosed antibodies mayspecifically bind to the BMP-11 protein. The BMP-11 protein isoptionally at least about 75%-80% identical to SEQ ID NO:16, optionallyat least about 81% to about 85% identical to SEQ ID NO:16, optionally atleast about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94% identical toSEQ ID NO:16, and optionally at least about 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID NO:16. The BMP-11 protein optionally comprisesSEQ ID NO:16.

In a particular embodiment, termed JA-16, the antibody comprises theamino acid sequence of SEQ ID NO: 1 as a part of the variable region ofthe heavy chain. In other embodiments, the antibody comprises at leastone single chain CDR chosen from the amino acids 30-35 of SEQ ID NO:1,amino acids 50-66 of SEQ ID NO:1, and amino acids 99-102 of SEQ ID NO:1.

One of skill in the art will recognize that the antibodies of theinvention may contain any number of conservative or non-conservativechanges to their respective amino acid sequences without altering theirbiological properties. Changes can be made in either the framework (FR)or in the CDR regions. While changes in the framework regions areusually designed to improve stability and immunogenicity of theantibody, changes in the CDRs are usually designed to increase affinityof the antibody for its target. Such affinity-increasing changes aretypically determined empirically by altering the CDR region and testingthe antibody. Such alterations can be made according to the methodsdescribed in Antibody Engineering, 2nd. ed., Oxford University Press,ed. Borrebaeck, 1995. Conservative amino acid modifications are based onthe relative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary conservative substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine, and isoleucine. Further details on such changes are describedin the following sections. Unlike in CDRs, more substantialnon-conservative changes in structure framework regions (FRs) can bemade without adversely affecting the binding properties of an antibody.Changes to FRs include, but are not limited to, humanizing a non-humanderived framework or engineering certain framework residues that areimportant for antigen contact or for stabilizing the binding site, e.g.,changing the class or subclass of the constant region, changing specificamino acid residues which might alter an effector function such as Fcreceptor binding (Lund et al. (1991) J. Immun. 147: 2657-2662 and Morganet al. (1995) Immunology 86: 319-324), or changing the species fromwhich the constant region is derived as described below.

In an embodiment, the presently disclosed antibodies specifically bindto mature GDF-8 protein, regardless of whether it is in monomeric form,active dimer form, or complexed in a GDF-8 latent complex, with anaffinity of between about 10⁶ and about 10¹¹ M⁻¹, optionally betweenabout 10⁸ and about 10¹¹ M⁻¹.

The antibodies of the present invention may comprise polyclonalantibodies, monoclonal antibodies, antibody compositions, antibodieshaving mono- or poly-specificity, humanized antibodies, single-chainantibodies, CDR-grafted antibodies, antibody fragments such as Fab,F(ab′)₂, Fv, and other antibody fragments which retain the antigenbinding function of the parent antibody. The presently disclosedantibodies may also be modified to chimeric antibodies. For instance, ahuman Fc region can be fused to a GDF-8 binding region from a murineantibody to generate a chimeric antibody. By replacing other portions ofthe murine antibody (outside of the antigen binding region) withcorresponding human antibody fragments, a humanized antibody may beproduced. Such chimeric or humanized antibodies may display enhancedbiological specificity or in vivo stability. They are particularlyuseful in designing antibodies for human therapies. It is understoodthat practitioners are familiar with the standard resource materialswhich describe specific conditions and procedures for the construction,manipulation, production, and isolation of antibodies (see, for example,Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y.).

The present invention also provides cells, such as hybridomas, thatproduce any of the presently disclosed antibodies. One of skill in theart is familiar with the many cells that are suitable for producingantibodies. Any cell compatible with the present invention may be usedto produce the presently disclosed antibodies. In an embodiment, thepresently disclosed antibodies are produced by a hybridoma cell. Ahybridoma cell line, which produces murine anti-GDF-8 JA-16 antibody hasbeen deposited with American Tissue Culture Collection (DepositDesignation Number PTA-4236) on Apr. 18, 2002. The address of thedepository is 10801 University Blvd, Manassas, Va. 20110.

Methods of Treating Disease

The antibodies of the present invention are useful to prevent, diagnose,or treat various medical disorders in humans or animals. The antibodiesare used to inhibit or reduce one or more activities associated with theGDF protein, relative to a GDF protein not bound by the same antibody.Optionally, the antibodies inhibit or reduce one or more of theactivities of mature GDF-8 (regardless of whether in monomeric form,active dimeric form, or complexed in a GDF-8 latent complex) relative toa mature GDF-8 protein that is not bound by the same antibodies. In anembodiment, the activity of the mature GDF-8 protein, when bound by oneor more of the presently disclosed antibodies, is inhibited at least50%, optionally at least 60, 62, 64, 66, 68, 70, 72, 72, 76, 78, 80, 82,84, 86, or 88%, optionally at least 90, 91, 92, 93, or 94%, andoptionally at least 95% to 100% relative to a mature GDF-8 protein thatis not bound by one or more of the presently disclosed antibodies.

The medical disorder being diagnosed, treated, or prevented by thepresently disclosed antibodies is optionally a muscle and neuromusculardisorder; an adipose tissue disorder such as obesity; type 2 diabetes,impaired glucose tolerance, metabolic syndromes (e.g., syndrome X),insulin resistance induced by trauma such as burns; or bone degenerativedisease such as osteoporosis. The medical condition is optionally amuscle or neuromuscular disorder, such as muscular dystrophy, muscleatrophy, congestive obstructive pulmonary disease, muscle wastingsyndrome, sarcopenia, or cachexia and disorders associated with a lossof bone, which include osteoporosis, especially in the elderly and/orpostmenopausal women, glucocorticoid-induced osteoporosis, osteopenia,and osteoporosis-related fractures. Other target metabolic bone diseasesand disorders amendable to treatment with GDF-8 antibodies of theinvention include low bone mass due to chronic glucocorticoid therapy,premature gonadal failure, androgen suppression, vitamin D deficiency,secondary hyperparathyroidism, nutritional deficiencies, and anorexianervosa. The antibodies are optionally used to prevent, diagnose, ortreat such medical disorders in mammals, optionally in humans.

The antibodies or antibody compositions of the present invention areadministered in therapeutically effective amounts. As used herein, an“effective amount” of the antibody is a dosage which is sufficient toreduce the activity of GDF proteins to achieve a desired biologicaloutcome (e.g., increasing muscle mass or strength). Generally, atherapeutically effective amount may vary with the subject's age,condition, and sex, as well as the severity of the medical condition inthe subject. The dosage may be determined by an physcian and adjusted,as necessary, to suit observed effects of the treatment. Generally, thecompositions are administered so that antibodies are given at a dosebetween 1 μg/kg and 20 mg/kg. Optionally, the antibodies are given as abolus dose, to maximize the circulating levels of antibodies for thegreatest length of time after the dose. Continuous infusion may also beused after the bolus dose.

The methods of treating, diagnosing, or preventing the above medicalconditions with the presently disclosed antibodies can also be used onother proteins in the TGF-β superfamily. Many of these proteins, e.g.,BMP-11, are related in structure to GDF-8. Accordingly, in anotherembodiment, the invention provides methods of treating theaforementioned disorders by administering to a subject an antibodycapable of inhibiting BMP-11 or activin, either alone or in combinationwith other TGF-β inhibitors, such as a neutralizing antibody againstGDF-8.

The antibodies of the present invention may be used to detect thepresence of proteins belonging to the TGF-β superfamily, such as BMP-11and GDF-8. By correlating the presence or level of these proteins with amedical condition, one of skill in the art can diagnose the associatedmedical condition. The medical conditions that may be diagnosed by thepresently disclosed antibodies are set forth above.

Such detection methods are well known in the art and include ELISA,radioimmunoassay, immunoblot, western blot, immunofluorescence,immuno-precipitation, and other comparable techniques. The antibodiesmay further be provided in a diagnostic kit that incorporates one ormore of these techniques to detect a protein (e.g., GDF-8). Such a kitmay contain other components, packaging, instructions, or other materialto aid the detection of the protein and use of the kit.

Where the antibodies are intended for diagnostic purposes, it may bedesirable to modify them, for example with a ligand group (such asbiotin) or a detectable marker group (such as a fluorescent group, aradioisotope or an enzyme). If desired, the antibodies (whetherpolyclonal or monoclonal) may be labeled using conventional techniques.Suitable labels include fluorophores, chromophores, radioactive atoms,electron-dense reagents, enzymes, and ligands having specific bindingpartners. Enzymes are typically detected by their activity. For example,horseradish peroxidase is usually detected by its ability to convert3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiablewith a spectrophotometer. Other suitable labels include, for example,one of the binding partners such as biotin and avidin or streptavidin,IgG and protein A, and various receptor-ligand couples known in the art.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art, and are considered as equivalents withinthe scope of the instant invention.

Antibody Compositions

The present invention provides compositions comprising the presentlydisclosed antibodies. Such compositions may be suitable forpharmaceutical use and administration to patients. The compositionstypically comprise one or more antibodies of the present invention and apharmaceutically acceptable excipient. As used herein, the phrase“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, that arecompatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.The compositions may also contain other active compounds providingsupplemental, additional, or enhanced therapeutic functions. Thepharmaceutical compositions may also be included in a container, pack,or dispenser together with instructions for administration.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. It may also be possible to obtain compositions which may betopically or orally administered, or which may be capable oftransmission across mucous membranes. The administration may, forexample, be intravenous, intraperitoneal, intramuscular, intracavity,subcutaneous or transdermal.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations may be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, isotonic agents,for example, sugars, polyalcohols such as manitol, sorbitol, sodiumchloride will be included in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theantibodies can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches, and the like cancontain any of the following ingredients, or compounds of a similarnature; a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the antibodies are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The antibodies may also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the presently disclosed antibodies are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscontaining the presently disclosed antibodies can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Antibodies which exhibit large therapeutic indices are an embodiment ofthe invention.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies optionally within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any antibody usedin the present invention, the therapeutically effective dose can beestimated initially from cell culture assays. A dose may be formulatedin animal models to achieve a circulating plasma concentration rangethat includes the IC₅₀ (i.e., the concentration of the test antibodywhich achieves a half-maximal inhibition of symptoms) as determined incell culture. Levels in plasma may be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by a suitable bioassay. Examples of suitable bioassaysinclude DNA replication assays, transcription-based assays, GDFprotein/receptor binding assays, creatine kinase assays, assays based onthe differentiation of pre-adipocytes, assays based on glucose uptake inadipocytes, and immunological assays.

Modified Antibodies

It is understood by one of ordinary skill in the art that certain aminoacids may be substituted for other amino acids in a protein structurewithout adversely affecting the activity of the protein, e.g., bindingcharacteristics of an antibody. It is thus contemplated by the inventorsthat various changes may be made in the amino acid sequences of thepresently disclosed antibodies, or DNA sequences encoding theantibodies, without appreciable loss of their biological utility oractivity. Such changes may include deletions, insertions, truncations,substitutions, fusions, shuffling of motif sequences, and the like.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle (1982) J. Mol. Biol., 157:105-132). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle,1982); these are isoleucine (+4.5), valine (+4.2), leucine (+3.8),phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9),alanine (+1.8), glycine (−0.4), threonine (−0.7), serine (−0.8),tryptophan (−0.9), tyrosine (−1.3), proline (−1.6), histidine (−3.2),glutamate (−3.5), glutamine (−3.5), aspartate (−3.5), asparagine (−3.5),lysine (−3.9), and arginine (−4.5).

In making such changes, the substitution of amino acids whosehydropathic indices are within ±2 is an embodiment of the invention,those which are within ±1 are optional, and those within ±0.5 are alsooptional.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as govern by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0),lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0±1), serine (+0.3),asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (−0.4),proline (−0.5±1), alanine (−0.5), histidine (−0.5), cysteine (−1.0),methionine (−1.3), valine (−1.5), leucine (−1.8), isoleucine (−1.8),tyrosine (−2.3), phenylalanine (−2.5), and tryptophan (−3.4).

In making such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is an embodiment of the invention,those within ±1 are optional, and those within ±0.5 are optional.

The modifications may be conservative such that the structure orbiological function of the protein is not affected by the change. Suchconservative amino acid modifications are based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplaryconservative substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. The amino acid sequence of the presently disclosedantibodies may be modified to have any number of conservative changes,so long as the binding of the antibody to its target antigen is notadversely affected. Such changes may be introduced inside or outside ofthe antigen binding portion of the antibody. For example, changesintroduced inside of the antigen binding portion of the antibody may bedesigned to increase the affinity of the antibody for its target.

In addition to the changes to the amino acid sequence outlined above,the antibodies can be glycosylated, pegylated, or linked to albumin or anonproteinaceous polymer. For instance, the presently disclosedantibodies may be linked to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antibodiesare chemically modified by covalent conjugation to a polymer to increasetheir circulating half-life, for example. Certain polymers, and methodsto attach them to peptides, are also shown in U.S. Pat. Nos. 4,766,106;4,179,337; 4,495,285; and 4,609,546.

In another embodiment, the antibody may be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used herein, “altered” means having one ormore carbohydrate moieties deleted, and/or having one or moreglycosylation sites added to the original antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the presently disclosed antibodies isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the sequence of the original antibodies (for O-linkedglycosylation sites). For ease, the antibody amino acid sequence isoptionally altered through changes at the DNA level.

Another means of increasing the number of carbohydrate moieties on theantibodies is by chemical or enzymatic coupling of glycosides to theamino acid residues of the antibody. These procedures are advantageousin that they do not require production of the GDF peptide inhibitor in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugars may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston (1981) CRC Crit. Rev.Biochem., 22: 259-306.

Removal of any carbohydrate moieties present on the antibodies may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to trifluoromethanesulfonic acid, oran equivalent compound. This treatment results in the cleavage of mostor all sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.

Chemical deglycosylation is described by Hakimuddin et al. (1987) Arch.Biochem. Biophys., 259: 52; and Edge et al. (1981) Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on GDF peptideinhibitors can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (I987) Meth. Enzymol.,138: 350.

Sequence Analysis

While not always necessary, if desired, one of ordinary skill in the artmay determine the amino acid or nucleic acid sequences of the presentlydisclosed antibodies. The present invention includes these amino acidand nucleic acid sequences. The present invention also include variants,homologues, and fragments of these nucleic and amino acid sequences. Forexample, the antibody may comprise a heavy chain variable regionsequence that comprises SEQ ID NO:1, or a nucleic acid sequence thatencodes SEQ ID NO:1 (e.g., SEQ ID NO:6). The nucleic or amino acidsequence optionally comprises a sequence at least 70% to 79% identicalto the nucleic or amino acid sequence of the presently disclosedvariable heavy chain region, optionally at least 80% to 89% identical,optionally at least 90% to 95% identical, and optionally at least 96% to100% identical. One of skill in the art will recognize that the CDRregion, which determines the antigenic binding properties of theantibody, can tolerate less sequence variation than the other portionsof the antibody not involved in antigen binding. Thus, these non-bindingregions of the antibody may contain substantial variations withoutsignificantly altering the binding properties of the antibody. However,one of skill in the art will also recognize that many changes can bemade to the CDR region that are specifically designed to increase theaffinity of the antibody for its target. Such affinity-increasingchanges are typically determined empirically by altering the CDR regionand testing the antibody. AU such alterations, whether within the CDR oroutside the CDR, are included in the scope of the present invention.

Relative sequence similarity or identity may be determined using the“Best Fit” or “Gap” programs of the Sequence Analysis Software Package™(Version 10; Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis.). “Gap” utilizes the algorithm ofNeedleman and Wunsch (Needleman and Wunsch, 1970) to find the alignmentof two sequences that maximizes the number of matches and minimizes thenumber of gaps. “BestFit” performs an optimal alignment of the bestsegment of similarity between two sequences. Optimal alignments arefound by inserting gaps to maximize the number of matches using thelocal homology algorithm of Smith and Waterman (Smith and Waterman,1981; Smith et al., 1983).

The Sequence Analysis Software Package described above contains a numberof other useful sequence analysis tools for identifying homologues ofthe presently disclosed nucleotide and amino acid sequences. Forexample, the “BLAST” program (Altschul et al., 1990) searches forsequences similar to a query sequence (either peptide or nucleic acid)in a specified database (e.g., sequence databases maintained at theNational Center for Biotechnology Information (NCBI) in Bethesda, Md.);“FastA” (Lipman and Pearson, 1985; see also Pearson and Lipman, 1988;Pearson et al., 1990) performs a Pearson and Lipman search forsimilarity between a query sequence and a group of sequences of the sametype (nucleic acid or protein); “TfastA” performs a Pearson and Lipmansearch for similarity between a protein query sequence and any group ofnucleotide sequences (it translates the nucleotide sequences in all sixreading frames before performing the comparison); “FastX” performs aPearson and Lipman search for similarity between a nucleotide querysequence and a group of protein sequences, taking frameshifts intoaccount. “TfastX” performs a Pearson and Lipman search for similaritybetween a protein query sequence and any group of nucleotide sequences,taking frameshifts into account (it translates both strands of thenucleic sequence before performing the comparison).

The following examples provide embodiments of the invention. One ofordinary skill in the art will recognize the numerous modifications andvariations that may be performed without altering the spirit or scope ofthe present invention. Such modifications and variations are believed tobe encompassed within the scope of the invention. The examples do not inany way limit the invention.

The entire contents of all references, patents and published patentapplications cited throughout this application are herein incorporatedby reference.

Examples Example 1 Purification of GDF-8

Conditioned media from a selected cell line expressing full-length humanGDF-8 protein (mature GDF-8+GDF-8 propeptide) were acidified to pH 6.5and applied to a 80×50 mm POROS HQ anion exchange column in tandem to a80×50 mm POROS SP cation exchange column (PerSeptive Biosystems, FosterCity, Calif.). The flow through was adjusted to pH 5.0 and applied to a75×20 mm POROS SP cation exchange column (PerSeptive Biosystems) andeluted with a NaCl gradient. Fractions containing the GDF-8, asindicated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), were pooled, acidified with trifluoroacetic acid (TFA) to pH2-3, then brought up to 200 ml with 0.1% TFA to lower the viscosity. Thepool was then applied to a 250×21.2 mm C₅ column (Phenomenex, Torrance,Calif.) preceded by a 60×21.2 mm guard column (Phenomenex) and elutedwith a TFA/CH₃CN gradient, to separate mature GDF-8 from GDF-8propeptide. Pooled fractions containing mature GDF-8 were concentratedby lyophilization to remove the acetonitrile and 20 ml of 0.1% TFA wasadded. The sample was then applied to a 250×10 mm C₅ column (Phenomenex)heated to 60° C. to aid in separation. This was repeated until furtherseparation could no longer be achieved. Fractions containing matureGDF-8 were then pooled and brought up to 40% acetonitrile and applied toa 600×21.2 BioSep S-3000 size exclusion column (Phenomenex) preceded bya 60×21.2 guard column. Fractions containing purified mature GDF-8 werepooled and concentrated for use in subsequent experiments.

C₅ column fractions containing GDF-8 propeptide were pooled, theacetonitrile was removed by evaporation, 20 ml of 0.1% TFA was added,and the sample was then injected onto the 250×10 mm C₅ column at 60° C.This was repeated until further separation could no longer be achieved.Fractions containing the GDF-8 propeptide were then pooled and broughtup to 40% acetonitrile and applied to a 600×21.2 BioSep S-3000 sizeexclusion column (Phenomenex) preceded by a 60×21.2 guard column.Fractions containing the purified GDF-8 propeptide were pooled andconcentrated for use in subsequent experiments.

On SDS-PAGE, purified mature GDF-8 migrated as a broad band at 25 kDaunder nonreducing conditions and 13 kDa under reducing conditions. Asimilar SDS-PAGE profile has been reported for murine GDF-8 (McPherronet al., 1997, supra), and reflects the dimeric nature of the matureprotein.

The apparent molecular weight of purified GDF-8 propeptide was 38 kDaunder both reducing and nonreducing conditions. This indicates that theGDF-8 propeptide by itself is monomeric. The difference between theapparent molecular weight and the predicted molecular weight of GDF-8propeptide, ˜26 kDa, may reflect the addition of carbohydrate, since itsamino acid sequence contains a potential N-linked glycosylation site(McPherron et al., 1997, supra).

Example 2 Characteristics of Purified Recombinant Human GDF-8

50 μg each of purified mature GDF-8 and purified GDF-8 propeptide weremixed and dialyzed into 50 mM sodium phosphate, pH 7.0, andchromatographed on a 300×7.8 mm BioSep S-3000 size exclusion column(Phenomenex). Molecular weight of the mature GDF-8:propeptide complexwas determined from elution time, using molecular weight standards(Bio-Rad Laboratories, Hercules, Calif.) chromatographed on the samecolumn.

When purified GDF-8 propeptide was incubated with purified mature GDF-8at neutral pH, the two proteins appeared to complex, as indicated by thesize exclusion profile. The primary protein peak eluted at 12.7 minuteshad an estimated molecular weight of 78 kDa from molecular weightstandards (Bio-Rad Laboratories, Hercules, Calif.) chromatographed onthe same column. The size of the complex is most consistent with onedimer of the mature GDF-8 associating with two monomers of propeptide.

To confirm this observation, a preparation containing both mature GDF-8and GDF-8 propeptide was incubated with or without 100 mM 1-Ethyl3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Pierce) for 1hour at room temperature (RT), acidified with HCl to pH 2-3, andconcentrated with a Micron-10 Amicon concentrator (Millipore, Bedford,Mass.) for SDS-PAGE, using a tricine buffered 10% acrylamide gel.Proteins were visualized by Coomassie blue staining of the gel. In thepresence of EDC, a cross-linked complex with an apparent molecularweight of 75 kDa was observed.

The GDF-8 propeptide DNA and amino acid sequence are set forth inMcPherron and Lee (1997) Proc. Natl. Acad. Sci. USA, 94: 12457-12461.

Example 3 Production of Anti-GDF-8 Antibody

To develop an antibody capable of inhibiting GDF-8 activity, a group ofGDF-8 knockout mice were immunized every two weeks with mature GDF-8protein (purified as described in Example 1) mixed in Freunds completeadjuvant for the first two immunizations, and incomplete Freundsadjuvant thereafter. Throughout the immunization period, blood wassampled and tested for the presence of circulating antibodies. At week9, an animal with circulating antibodies was selected, immunized forthree consecutive days, and sacrificed. The spleen was removed andhomogenized into cells. The spleen cells were fused to a myeloma fusionpartner (line P3-x63-Ag8.653) using 50% PEG 1500 by an establishedprocedure (Oi & Herzenberg (1980) Selected Methods in CellularImmunology, W. J. Freeman Co., San Francisco, Calif., p. 351). The fusedcells were plated into 96-well microtiter plates at a density of 2×10⁵cells/well. After 24 hours, the cells were subjected to HAT selection(Littlefield (1964) Science, 145: 709) effectively killing any unfusedand unproductively fused myeloma cells.

Successfully fused hybridoma cells secreting anti-GDF-8 antibodies wereidentified by solid and solution phase ELISAs. Mature GDF-8 protein wasprepared from CHO cells as described above and coated on polystyrene(for solid phase assays) or biotinylated (for a solution based assay).Neutralizing assays were also employed where the ActRIIB receptor wascoated on a polystyrene plate and biotin GDF-8 binding was inhibited bythe addition of hybridoma supernatant. Results identified hybridomasexpressing GDF-8 antibodies. These positive clones were cultured andexpanded for further study. These cultures remained stable when expandedand cell lines were cloned by limiting dilution and cryopreserved.

From these cell cultures, a panel of antibodies was developed thatspecifically recognize mature GDF-8. Isotype of the antibodies wasdetermined using a mouse immunoglobulin isotyping kit (ZymedLaboratories, San Francisco, Calif.). One of the antibody clones,designated JA-16, was studied further.

Example 4 Characterization of JA-16 Binding Specificity

To determine the binding specificity of JA-16, a panel of syntheticpeptides corresponding to portions of the GDF-8 protein sequence wasproduced. FIG. 1 shows the GDF-8 synthetic peptides used in this study.Even number peptides (N2-N14) were biotinylated on the primary amine.The biotinylated peptides N2, N4, N6, N8, N10, N12, N14, and anirrelevant peptide DAE-10, were coated at 1 μg/ml for 2 hrs at roomtemperature on ReactiBind™ Streptavidin coated polystyrene 96 wellplates (Pierce, Rockford, Ill., Cat. No. 15124) following themanufacturer's protocol.

After blocking, JA-16 or a unrelated monoclonal antibody control wasadded to the ELISA plate at 100, 10, and 1 nM (JA-16 only), andincubated for 30 min. After washing the plate, a secondary antibody(goat anti-murine IgG (H+L)-HRP, Calbiochem, San Diego, Calif., Cat. No.401215) was added at a 1:1000 dilution and incubated for 30 minutes atroom temperature. The plate was washed four times, and TMB substrate wasadded (KPL, Gaithersburg, MD, Cat. No. 50-76-04). Colorimetricmeasurements were done at 450 nm in a Molecular Devices microplatereader. The results are shown in FIG. 2. JA-16 bound strongly andspecifically to the biotinylated N-terminal peptide N8 (SEQ ID NO:65).

Mature GDF-8 and BMP-11 are 90% homologous at the amino acid level (FIG.3). Three of these changes are present within the N8 peptide. To comparethe specificity of JA-16 towards GDF-8 and BMP-11, shorter peptides weredesigned G1 and B1 specific for GDF-8 and BMP-11, respectively.Differences between G1 and B1 are indicated with underlining.

G1: (SEQ ID NO: 10) Asp-Phe-Gly-Leu-Asp-Ser-Asp-Glu-His-Ser-Thr-Glu-Ser-Arg-Cys B1: (SEQ ID NO: 9)Asn-Leu-Gly-Leu-Asp-Ser-Asp-Glu-His-Ser-Ser-Glu- Ser-Arg-Cys

The peptides G1 and B1 were conjugated to BSA using a PIERCE conjugationkit (Cat. No. 77116ZZ) following manufacturer's protocol. G1-BSA andB1-BSA were coated on 96 well flat-bottom assay plates (Costar, N.Y.,Cat. No. 3590) at 1 μg/ml in 0.2 M sodium carbonate buffer overnight at4° C. The plates were washed and blocked with PBS, 1 mg/ml BSA, 0.05%Tween for 1 hour at room temperature. JA-16 (5 nM) was serially diluted(1:2). The dilutions were added to the ELISA plate and incubated for 30min at RT. After 4 washes, a secondary antibody (goat anti-murine IgG(H+L)-HRP, Calbiochem, Cat. No. 401215) was added at a 1:1000 dilutionand incubated for 30 min at RT. Plates were washed four times, and TMBsubstrate was added (KPL, Cat. No. 50-76-04). Colorimetric measurementswere done at 450 nm in a Molecular Devices microplate reader. FIG. 4shows that JA-16 binds to G1-BSA in a concentration dependent manner,but not to B1-BSA, even at the highest concentration.

To look further into JA-16 specificity, G1-BSA was coated as describedabove, but this time, JA-16 at 5 nM was preincubated with either G1peptide or B1 peptide, GDF-8, or BMP-11 at various concentrations. Theresult is shown in FIG. 5. The BMP-11 specific peptide B1 does notinhibit binding of JA-16 to G1-BSA, but G1 does. The IC₅₀ for GDF-8 is0.8 μg/ml, while BMP-11's IC₅₀ is 3.8 μg/ml demonstrating that JA-16recognizes GDF-8 with a 5-fold higher affinity than BMP-11.

Example 5 Mapping of JA-16 Epitope

In order to map the exact epitope of JA-16, overlapping 13-mer peptides(SEQ ID NOS:17-64, see FIG. 6A) corresponding to portions of the GDF-8sequence were synthesized directly on cellulose paper using the spotsynthesis technique (Molina et al. (1996) Peptide Research, 9: 151-155;Frank et al. (1992) Tetrahedron, 48: 9217-9232). In this array, cysteineresidues were replaced with serine in order to reduce the chemicalcomplications that are caused by the presence of cysteines. Cellulosemembranes modified with polyethylene glycol and Fmoc-protected aminoacids were purchased from Abimed (Lagenfeld, Germany). The array wasdefined on the membrane by coupling a β-alanine spacer and peptides weresynthesized using standard DIC (diisopropylcarbodiimide)/HOBt(hydroxybenzotriazole) coupling chemistry as described previously(Molina et al. (1996) Peptide Research, 9: 151-155; Frank et al. (1992)Tetrahedron, 48: 9217-9232).

Activated amino acids were spotted using an Abimed ASP 222 robot.Washing and deprotection steps were done manually and the peptides wereN-terminally acetylated after the final synthesis cycle. Followingpeptide synthesis, the membrane was washed in methanol for 10 minutesand in blocker (TBST (Tris buffered saline with 0.1% (v/v) Tween 20)+1%(w/v) casein) for 10 minutes. The membrane was then incubated with 2.5μg/ml JA-16 in blocker for 1 hour with gentle shaking. After washingwith blocker 3 times for 10 minutes, the membrane was incubated withHRP-labeled secondary antibody (0.25 μg/ml in blocker) for 30 minutes.The membrane was then washed 3 times for 10 minutes each with blockerand 2 times for 10 minutes each with TBST. Bound antibody was visualizedusing SuperSignal West reagent (Pierce) and a digital camera(AlphaInnotech FluorImager). Results are shown in FIG. 6B. JA-16 boundto the first 4 peptides of the array (SEQ ID NOS:17-20), whichcorresponds to 18 residues on the N-terminus of GDF-8.

In order to further characterize the JA-16 epitope, deletion andsubstitution analyses of the peptideGly-Leu-Asp-Ser-Asp-Glu-His-Ser-Thr-Glu-Ser-Arg-Ser (SEQ ID NO:18) wereperformed using spot synthesis. In the substitution analysis, eachresidue of this peptide was individually replaced with each of the 20natural amino acids except cysteine, generating SEQ ID NOS: 3, 18,66-104, 106-113, and 115-128. Synthesis and binding assays wereperformed as described above. The results are shown in FIG. 7.Substitutions in the 4 N-terminal amino acids and the 4 C-terminal aminoacids were well tolerated, suggesting that these amino acids were notneeded for JA-16 binding to mature GDF-8. However, no changes weretolerated in the middle segment of this peptide, Asp-Glu-His-Ser-Thr(SEQ ID NO:3), except for a few substitutions at the serine residue,suggesting that this peptide sequence was required for JA-16 binding. Inaddition, the sequence Asp-Glu-His-Ser-Thr (SEQ ID NO:3) was thesmallest peptide to which binding could be detected in the deletionanalysis. Thus, the results suggest that JA-16 recognizes the epitopeAsp-Glu-His-Ser-Thr (SEQ ID NO:3) in GDF-8, with the Asp, Glu, His andThr residues (Asp-Glu-His-Xaa-Thr (SEQ ID NO:2)) being important forbinding.

Example 6 Characterization of JA-16 In Vitro

Two assays were performed to determine the ability of JA-16 toneutralize GDF-8 activity in vitro. First, JA-16 was tested for itsability to inhibit mature GDF-8 protein binding to the ActRIIB receptor.Recombinant ActRIIB.Fc chimera (R&D Systems, Minneapolis, Minn., Cat.No. 339-RB/CF) was coated on 96 well flat-bottom assay plates (Costar,Cat. No. 3590) at 1 μg/ml in 0.2 M sodium carbonate buffer overnight at4° C. Plates were then blocked with 1 mg/ml bovine serum albumin andwashed following standard ELISA techniques.

100 μl of biotinylated mature GDF-8 protein at various concentrationswas added to the blocked ELISA plates, incubated for 1 hour, and washed.The amount of bound mature GDF-8 protein was detected bystreptavidin-horseradish peroxidase (SA-HRP, BD PharMingen, San Diego,Calif., Cat. No. 13047E) followed by the addition of TMB (KPL, Cat. No.50-76-04). Colorimetric measurements were done at 450 nm in a MolecularDevices microplate reader. The results are shown in FIG. 8. The matureGDF-8 exhibited an ED₅₀ of 12 ng/ml.

The same protocol was also performed after preincubating the JA-16antibody with biotinylated mature GDF-8 protein at 5 ng/ml for 30 min.An irrelevelant monoclonal antibody was included as a negative control.FIG. 9 shows that JA-16 has a very weak in vitro neutralizing activityof around 1 μM. This in vitro data suggests that JA-16 is unlikely to bea very good neutralizer of active GDF-8, particularly under lesscontrolled in vivo conditions.

In a second set of assays, a reporter gene assay was performed to assessthe biological activity of active GDF-8 protein in vitro. The assay usesa reporter vector, pGL3(CAGA)₁₂, coupled to luciferase. The CAGAsequence was previously reported to be a TGF-β-responsive sequencewithin the promoter of the TGF-β-induced gene, PAI-1.

The reporter vector containing 12 CAGA boxes was made using the basicreporter plasmid, pGL3 (Promega Corporation, Madison, Wis., Cat. No.E1751). The TATA box and transcription initiation site from theadenovirus major late promoter (−35/+10) was inserted between the BgIIIand HindIII sites. Oligonucleotides containing twelve repeats of theCAGA boxes AGCCAGACA were annealed and cloned into the Xhol site. Thehuman rhabdomyosarcoma cell line, A204 (ATCC HTB-82), was transientlytransfected with pGL3(CAGA)₁₂ using FuGENE 6 transfection reagent (RocheDiagnostics, Indianapolis, Minn. Cat. No. 1 814 443). Followingtransfection, cells were cultured on 48 well plates in McCoys 5A medium(Life Technologies, Rockville, Md., Cat. No. 21500-079) supplementedwith 2 mM glutamine, 100 U/ml streptomycin, 100 μg/ml penicillin and 10%fetal calf serum for 16 h. Cells were then treated with mature GDF-8,BMP-11, or activin in McCoy's 5A media with glutamine, streptomycin,penicillin, and 1 mg/ml bovine serum albumin for 6 h at 37° C.Luciferase was quantified in the treated cells using the LuciferaseAssay System (Promega Corporation, Madison, Wis., Cat. No. E1483). GDF-8maximally activated the reporter construct 10-fold, with an ED₅₀ of 10ng/ml GDF-8. BMP-11, which is 90% identical to GDF-8 at the amino acidlevel (Gamer et al. (1999) Dev. Biol., 208(1): 222-32; Nakashima et al.(1999) Mech. Dev., 80(2): 185-9), and activin elicited a similarbiological response.

JA-16's neutralizing activity was determined by preincubating JA-16 withmature GDF-8 protein for 30 min prior to addition to the A204 cells. Anirrelevant antibody (monoclonal control) as well as a human GDF-8antibody derived from scFv phagemid library using phage displaytechnology (Myo-19) were also tested.

FIG. 10 shows that, in this assay as well, JA-16 is weakly neutralizingwith an IC₅₀ of around 1 μM, while the Myo-19 IC₅₀ is around 100 nM.Based on this in vitro data, one would have expected the Myo-19 antibodyto be a better neutralizer of active GDF-8 protein than JA-16 in vivo,which is not the case, as shown herein.

Example 7 Immunoprecipitation of GDF-8 with JA-16

In order to evaluate the binding of JA-16 to mature GDF-8 and GDF-8complexes, a series of immunoprecipitation studies were conducted.

First, to determine if JA-16 can immunoprecipitate the GDF-8 latentcomplex, CHO cells expressing GDF-8 were radiolabeled with³⁵S-methionine and ³⁵S-cysteine. 100 μl of conditioned medium from thesecells containing GDF-8 latent complex was incubated with 1 mg/ml JA-16for 1 hour at 4° C. Protein A Sepharose was added to the mixture, whichwas then incubated overnight at 4° C. The immunoprecipitate wascollected, washed three times with a PBS/Triton-X100 buffer, resuspendedin reducing sample buffer and analyzed by SDS-PAGE. The gel was fixedovernight, enhanced with autoradiography enhancer solution, dried andthe autoradiogram was developed. FIG. 18, lane 2, shows that JA-16 canimmunoprecipitate the GDF-8 latent complex and unprocessed GDF-8.

Second, to determine if JA-16 can immunoprecipitate a complex formedbetween GDF-8 and follistatin, CHO cells expressing follistatin wereradiolabeled with ³⁵S-methionine and ³⁵S-cysteine. 100 μl of conditionedmedium containing radiolabeled follistatin was mixed with mature GDF-8to form a complex of GDF-8 with follistatin. The mixture was incubatedwith 1 mg/ml JA-16 for 1 hour at 4° C. Protein A Sepharose was added tothe mixture, which was then incubated overnight at 4° C. Theimmunoprecipitate was collected and analyzed as described above. FIG.18, lane 6, shows that JA-16 can co-immunoprecipitate labeledfollistatin complexed with GDF-8.

Third, to investigate whether JA-16 can immunoprecipitate mature GDF-8protein, conditioned media from CHO cells containing radiolabeled GDF-8latent complex was acid activated to dissociate the GDF-8 propeptide andmature GDF-8 (see van Waarde et al. (1997) Analytical Biochemistry, 247,45-51). This material was then incubated with JA-16 for 1 hour at 4° C.The remainder of the protocol was performed as described above. FIG. 18,lane 3, shows that JA-16 can immunoprecipitate mature GDF-8.

The results indicate that JA-16 can recognize the GDF-8 latent complex,the GDF-8:follistatin complex, and mature GDF-8. In contrast, Myo-19cannot bind any GDF-8 complexes (FIG. 18, lanes 4 and 7) and can onlyimmunoprecipitate mature GDF-8 (FIG. 18, lane 5).

Example 8 Characterization of JA-16 In Vivo

In order to determine if the antibody JA-16 increases muscle mass inadult mice, an in vivo study was conducted with seven-week-old femaleBALB/c mice. Mice were weighed and evenly distributed with respect tobody weight into groups of seven or eight. JA-16 in PBS or an isotypematched antibody to snake venom (control) was injected into the miceintraperitoneally at 50 mg/kg twice weekly. The treatment continued forfour weeks. Animals were assessed for gain in lean body mass bysubjecting them to dexascan analysis before and after the treatmentperiod. Muscle mass was assessed by dissecting and weighing thegastrocnemius and quadriceps. The peri-uterine fat pad was also removedand weighed. The results of this study indicated that JA-16significantly inhibits GDF-8 activity in vivo resulting in increasedmuscle mass (FIG. 11).

A longer study was also performed in which the antibodies wereadministered intraperitoneally at 60 mg/kg/week for 14 weeks. These micewere loaded at the beginning of the study with 60 mg/kgintraperitoneally and 10 mg/kg intravenously. The mice in this studywere male C57BL mice that were either wild type at the agouti locus (a)or carried the lethal yellow mutation (Ay) at that locus. The Aymutation causes adult onset obesity and diabetes, which allowed us todetermine the effect of JA-16 on muscle, excess fat, and blood glucosein a diabetic background. Total body mass was measured weekly (FIG. 12).Muscle mass was assessed by dissecting and weighing the gastrocnemiusand quadriceps (FIG. 13). The epididymal and inguinal fat pads were alsoremoved and weighed (FIG. 14). Twelve weeks into the study, the micewere fasted and blood glucose levels were measured (FIG. 15). As withthe four week study, the results of this study indicate that JA-16inhibits GDF-8 activity in vivo causing an increase in muscle mass. Inaddition, this study indicates that in obese and diabetic mice,inhibition of GDF-8 leads to improved levels of blood glucose.

The in vivo activity of JA-16 was also compared to the in vivo activityof another GDF-8 antibody, Myo-19. C57B6/scid mice we injectedintraperitoneally for five weeks with vehicle control or with 60 mg/kgloading dose plus 60 mg/kg per week of JA-16 or Myo-19. Total body masswas measured weekly and muscle mass was assessed by dissecting andweighing the gastrocnemius and quadriceps (FIG. 17). While five weeks oftreatment with JA-16 led to an increase in muscle mass, treatment withMyo-19 did not effect muscle mass. In another experiment, Myo-19treatment was extended to 10 and to 15 weeks, and no increase in bodymass or muscle mass was seen for these time points.

Thus, despite the fact that the in vitro data suggested that JA-16 was aweaker neutralizer than Myo-19, the mouse studies clearly, butunexpectedly, demonstrate that JA-16 effectively reduces GDF-8 activityin vivo while Myo-19 does not. These results indicate that the specificsite on GDF-8 to which JA-16 binds is unique in that this site isresponsible for the formation of a stable inhibitory GDF-8:antibodycomplex in vivo. Thus, it is expected that any antibody specificallybinding site, as identified in Example 4, will possess in vivoneutralizing properties similar to or better than JA-16.

Example 9 JA-16 Increases Muscle Strength

In humans, muscle size and strength decreases by approximately 1% peryear starting in the third decade of life. For many aged people, theloss in muscle mass is significantly debilitating. This condition isknown as sarcopenia, or age related loss of muscle. In order todetermine if anti-GDF-8 treatment is effective for sarcopenia, aged mice(19 months of age at the beginning of the study and 21 months of age atthe end of the study) were treated with JA-16 for 8 weeks at 60 mg/kgonce a week. In the same experiment, young mice (2 month of age at thebeginning of the study and 4 months of age at the end of the study) weretreated with the same dose of JA-16. At the end of the study, bothgroups of mice had greater muscle mass than the vehicle treated controlsas seen, for example, from the quadriceps mass comparison (FIG. 19A).

In order to confirm that the increase in muscle size leads to anincrease in muscle strength, we performed grip strength tests with agedand young mice treated with JA-16 for eight weeks using a meterpurchased from Columbia Instruments (Columbus, Ohio; model 1027csx).Mice were allowed to grip and pull on the grid, and the peak force ofthe pull was recorded. Untrained mice were tested five times insuccession without rest. The peak force for each test was recorded andthe results of the five tests were averaged for each mouse. After sevenweeks of treatment, the peak force for the young JA-16 treated mice was10% greater and for the aged JA-16 treated mice was 13% greater than thepeak force for vehicle treated mice (FIG. 19B). In addition,longitudinal measurements taken before and after 7 weeks of treatmentshowed that strength of the aged mice increased by 17% (p<0.01) withJA-16 treatment, while the strength of the vehicle treated aged mice wasnot significantly changed (3.3%, p=0.66). These results confirm thatGDF-8 inhibition leads to an increase in muscle size and strength inboth young and aged mice and that it may be a useful therapy forsarcopenia.

Example 10 JA-16 Increases Muscle Mass and Strength in Dystrophic Muscle

The ability of in vivo inhibition of GDF-8 to ameliorate musculardystrophy was tested in the mdx mouse model of Duchenne's musculardystrophy (DMD). The DMD model has been described, for example, byTorres et al. (Brain (1987) 110, 269-299) and Hoffman et al. (Science(1987) 238, 347-350).

Four week old male mdx mice were treated with weekly intraperitonealinjections of JA-16 (60 mg/kg), and vehicle alone (control group) for 3months. To quantify the increase of muscle mass, animals were sacrificedand extensor digitorum longus (EDL) muscles dissected out and weighed.As shown in FIG. 20A, EDL muscles from the treated group of animalsweighed significantly more than controls. Of note, the relative increasein muscle mass was greater than the increase in body weight as shown inFIG. 20B. Consistently with this data, other muscle groups including thegastrocnemius, tibialis anterior and quadriceps were found to havesimilar increases in weight.

To quantify the absolute force production or muscle strength, werecorded the maximal isometric force produced upon depolarization ofmuscle using field electrodes. FIGS. 20C and 20D show that the JA-16treated mdx mice were able to exert a significantly higher maximal forceduring either twitch or tetanus. The increase in muscle strength wasproportional to the increase in muscle mass (FIGS. 20A, 20C, and 20D).These results offer physiological evidence for predicted therapeuticefficacy of GDF-8 inhibitors such as JA-16 in treatment of musculardystrophy and related diseases.

To independently verify the amelioration of the dystrophic phenotypeobserved in the mdx diaphragms, as well as ascertain improvement in thepathological status of mdx skeletal musculature in toto, we analyzedserum Creatine kinase (CK) levels from these mice. Extremely high levelsof CK are consistently noted with dystrophin-deficiency in mdx mice andhumans due to sarcolemmal damage (Bulfield et al. (1984) Proc. Natl.Acad. Sci. USA 81, 1189-1192 and Matsuda et al. (1995) J. Biochem.(Tokyo) 118, 959-64). At the start of the trial both the treated andcontrol groups of mdx mice had marked elevations of serum CK compared tonormal mice. However, after three months of in vivo myostatin blockadethere was a dramatic decline in serum CK levels of treated mdx mice(FIG. 4 c). The decrease in muscle degeneration and fibrosis coupledwith reduction of CK offers histological and biochemical evidence for afunctional improvement in mdx muscle produced by myostatin blockade invivo.

Example 11 In Vivo Role of GDF-8 in Trabecular Bone

Increased mechanical loading, either due to increased muscle activity orincreased body weight, is associated with increased bone mass and bonedensity. Therefore, GDF-8 knockout (KO) mice were assessed for alteredbone mass and microarchitecture. An initial assessment of adult miceshowed that bone density in the spine of the KO mice was nearly two-foldhigher than that of their wild-type littermates. This increase farexceeded what might have been expected to be solely due to the increasedmuscle mass in the GDF-8 KO mice.

High resolution microtomographic imaging (μCT40, Scanco Medical,Switzerland) was used to assess the trabecular bone volume fraction andmicroarchitecture in the 5th lumbar vertebrae and distal femora andcortical bone geometry at the femoral mid-diaphysis of adult GDF-8wildtype (WT) and KO mice. Specimens were taken from 9-10 month oldGDF-8 male and female KO and littermate controls (four mice of eachgenotype and sex). The entire vertebral body and femur were scannedusing microcomputed tomography at 12 μm resolution. Regions of interestencompassing the trabecular bone of the vertebral body or the trabecularbone of the distal femoral metaphysis (i.e., secondary spongiosa) wereidentified using a semi-automated contouring algorithm. The followingparameters were computed using direct 3D assessments: bone volumefraction (%), trabecular thickness (μm), separation (μm) and number(1/mm). In addition, the connectivity density, an indicator of how wellthe trabecular network is connected, was assessed as well as corticalbone parameters at the middiaphyseal region in the femur, includingtotal area, bone area, and cortical thickness.

Both male and female KO mice had dramatically increased trabecular bonedensity in the vertebral body compared to WT littermates (n=4, +93% and+70%, respectively, p<0.0001). This increased trabecular bone densitywas accompanied by a 14% increase in trabecular thickness (p=0.03), a38% increase in trabecular number (p=0.0002), and a 10% decrease intrabecular separation (p=0.009). The combined effect of these changes inarchitecture and density resulted in a 3.4- and 1.7-fold increase inconnectivity in male and female KO, respectively, compared to their WTlittermates (p<0.0001). In addition, a rough measure of the level ofmineralization of the trabecular bone indicated that the average mineralcontent of the trabeculae was 8% higher in the KO mice relative to thecontrols (p<0.0001). There is a hint that the effect is larger in malethan female mice, but the sample size is too small to make definitiveconclusions. Vertebral trabecular bone characteristics assessed byhigh-resolution microcomputed tomography are shown in Table 1.

In contrast to observations in the spine, male and female KO mice hadlower trabecular bone density in the distal femur than WT littermates(n=4, p=0.05 for overall genotype effect) (Table 2). This decrement inbone density was more pronounced in female KO than in male KO mice.GDF-8 KO mice had similar trabecular thickness as their WT littermates,but had fewer trabeculae and increased trabecular separation compared tolittermate controls. However, although cortical thickness at the femoralmidshaft was similar in male GDF-8 KO and their littermate controls, itwas approximately 10% greater in the GDF-8 KO female mice than their WTlittermates (n=4, p=0.04) (see Table 3). There were no differences incortical bone area or bone area fraction between the two genotypes.

TABLE 1 Vertebral Trabecular Bone Characteristics (mean ± SEM) Male WTMale KO Female WT Female KO Bone volume 23.3 ± 4.7 45.0 ± 5.5  27.5 ±5.5 46.9 ± 10.8 fraction (%) Trabecular 52 ± 3 58 ± 6  52 ± 5 61 ± 8 thickness (μm) Trabecular 210 ± 21 145 ± 8  183 ± 21 169 ± 41 separation (μm) Trabecular  4.6 ± 0.4 7.0 ± 0.4  5.2 ± 0.4 6.6 ± 1.3number (1/mm) Connectivity 137 ± 15 470 ± 114 198 ± 29 339 ± 81  density(1/mm³) Degree of  1.68 ± 0.08 1.29 ± 0.02  1.54 ± 0.12 1.34 ± 0.03anisotropy

TABLE 2 Characteristics of the Trabecular Bone in Distal FemoralMetaphysis (mean ± SEM) Male WT Male KO Female WT Female KO Bone volume5.1 ± 1.8 2.9 ± 1.7 11.9 ± 7.0 5.4 ± 3.1 fraction (%) Trabecular  68 ±1.2  68 ± 2.7 73 ± 7 63 ± 9  thickness (μm) Trabecular 353 ± 16  472 ±90  296 ± 73 464 ± 98  separation (μm) Trabecular 2.84 ± 0.12 2.24 ±0.51  3.46 ± 0.69 2.26 ± 0.57 number (1/mm) Connectivity 5.9 ± 3.7 4.0 ±6.9  31.5 ± 25.2 15.4 ± 15.1 density (1/mm³)

TABLE 3 Characteristics of the Cortical Bone at the FemoralMid-Diaphysis (mean ± SEM) Male WT Male KO Female WT Female KO Bone Area(mm²) 5.1 ± 1.8 2.9 ± 1.7 11.9 ± 7.0  5.4 ± 3.1 Cortical Thickness  68 ±1.2  68 ± 2.7 73 ± 7 63 ± 9 (μm) Bone Area/Total 353 ± 16  472 ± 90  296± 73 464 ± 98 Area (%)

Example 12 Treatment of Muscle and Bone Degenerative Disorders

Inhibitors of GDF-8, such as, for example inhibitory antibodies, areuseful for treatments directed at increased muscle mass, and also forprevention and treatment of osteoporosis. In addition, inhibition ofGDF-8 may be useful in other instances where a bone anabolic effect isdesired, such as augmentation of bone healing (i.e., fracture repair,spine fusion, etc.). The anti-GDF-8 antibodies of the invention are usedto treat a subject at disease onset or having an established muscle orbone degenerative disease.

Efficacy of anti-GDF-8 antibodies for treatment of bone disorders, e.g.,osteoporosis, is confirmed using well established models ofosteoporosis. For example, ovariectomized mice have been used to testthe efficacy of new osteoporosis drug treatments (Alexander et al.(2001) J. Bone Min. Res. 16: 1665-1673; and Anderson et al. (2001) J.Endocrinol. 170:529-537). Similar to humans, these rodents exhibit arapid loss of bone following ovariectomy, especially in cancellous bone.Outcome assessments are based on bone mineral density, biochemicalmarkers of bone turnover in serum and urine, bone strength, andhistology/histomorphometry.

In one study, normal and/or immune compromised female mice areovariectomized at 12-16 weeks of age and allowed to lose bone for fourto six weeks. Following this bone loss period, treatment with ananti-GDF-8 antibody such as JA-16 (IP injection) or vehicle is conductedfor one to six months. The treatment protocol could vary, with testingof different doses and treatment regimens (e.g., daily, weekly, orbi-weekly injections). It is anticipated that untreated ovariectomizedmice (or rats) would lose approximately 10-30% of bone density relativeto intact (i.e., non-ovariectomized), age-matched mice. It isanticipated that mice treated with the anti-GDF-8 antibody would have 10to 50% greater bone mass and bone density than those mice receivingplacebo, and moreover that this increase in bone density would beassociated with increased bone strength, particularly in regions with agreater proportion of cancellous bone compared to cortical bone.

The goal of another study is to demonstrate that anti-GDF-8 antibodysuch as JA-16 is effective in preventing the decline in bone mass,microarchitecture and strength associated with estrogen deficiency.Thus, the study has a similar design to the one described above, exceptthat treatment with anti-GDF-8 antibody would be initiated immediatelyafter ovariectomy, rather than after the bone loss period. It isanticipated that mice treated with the antibody would lose significantlyless bone mass following ovariectomy than mice treated with vehicle.

The inhibitory antibodies against GDF-8 are also used to prevent and/orto reduce severity and/or the symptoms of the disease. It is anticipatedthat the anti-GDF-8 antibodies would be administered as a subcutaneousinjection as frequently as once per day and as infrequently as once permonth. Treatment duration could range between one month and severalyears.

To test the clinical efficacy of anti-GDF-8 in humans, postmenopausalwomen with low bone mass are identified by bone density testing andrandomized to a treatment group. Treatment groups include a placebogroup and one to three groups receiving antibody (different doses).Individuals are followed prospectively for one to three years to assesschanges in biochemical markers of bone turnover, changes in bone mineraldensity, and the occurrence of fragility fractures. It is anticipatedthat individuals receiving treatment would exhibit an increase in bonemineral density in the proximal femur and lumbar spine of 2-30% relativeto baseline, and would have a decreased incidence of fragilityfractures. It is anticipated that biochemical markers of bone formationwould increase.

The antibodies are administered as the sole active compound or incombination with another compound or composition. When administered asthe sole active compound or in combination with another compound orcomposition, the dosage may be between approximately 1 μg/kg and 20mg/kg, depending on the severity of the symptoms and the progression ofthe disease. The appropriate effective dose is selected by a treatingclinician from the following ranges: 1 μg/kg and 20 mg/kg, 1 μg/kg and10 mg/kg, 1 μg/kg and 1 mg/kg, 10 μg/kg and 1 mg/kg, 10 μg/kg and 100μg/kg, 100 μg and 1 mg/kg, and 500 μg/kg and 1 mg/kg. Exemplarytreatment regimens and outcomes are summarized in Table 4.

TABLE 4 Examples of Clinical Cases Status prior to Treatment Patient No.treatment Regimen Outcome Patient 1 No clinical 0.01-1 mg/kg Maintenancesigns, biweekly for and/or increase postmenopausal 4-24 weeks ofmuscle/bone and/or over 60 mass years old Patient 2 Mild clinical0.01-20 mg/kg Maintenance signs, muscle weekly for 4 and/or increasewasting and/or more weeks of muscle/bone bone loss mass Patient 3Advanced stage 0.01-20 mg/kg Improvement of of osteoporosis twice weeklyfor clinical signs, 6 or more maintenance weeks and/or increase ofmuscle/bone mass Patient 4 Severe muscle 0.01-20 mg/kg Improvement ofand bone loss daily for 6 or clinical signs, more weeks reduction inseverity of symptoms and/or increase of muscle/bone mass

Example 13 Treatment of Metabolic Disorders

Inhibitors of GDF-8, such as, for example inhibitory antibodies, areuseful for treatment of metabolic disorders such as type 2 diabetes,impaired glucose tolerance, metabolic syndrome (e.g., syndrome X),insulin resistance induced by trauma (e.g., burns), and adipose tissuedisorders (e.g., obesity). The anti-GDF-8 antibodies of the inventionare used to treat a subject at disease onset or having an establishedmetabolic disease.

Efficacy of anti-GDF-8 antibodies for treatment of metabolic disorders,e.g., type 2 diabetes and/or obesity, is confirmed using wellestablished murine models of obesity, insulin resistance and type 2diabetes, including ob/ob, db/db, and strains carrying the lethal yellowmutation. Insulin resistance can also be induced by high fat or highcaloric feeding of certain strains of mice including, C57BL/6J. Similarto humans, these rodents develop insulin resistance, hyperinsuliemia,dyslipidemia, and deterioration of glucose homeostasis resulting inhyperglycemia. Outcome assessments are based on serum measurements ofglucose, insulin, and lipids. Improved insulin sensitivity can bedetermined by insulin tolerance tests and glucose tolerance tests. Moresensitive techniques would include the use ofeuglycemic-hyperinsulinemic clamps for assessing improvements isglycemic control and insulin sensitivity. In addition, the clamptechniques would allow a quantitative assessment of the role of themajor glucose disposing tissues (e.g., muscle, adipose, and liver) inimproved glycemic control.

In one study, treatment with an anti-GDF-8 antibody such as JA-16 (IPinjection) or vehicle is conducted for one week to six months. Thetreatment protocol could vary, with testing of different doses andtreatment regimens (e.g., daily, weekly, or bi-weekly injections). It isanticipated that mice treated with the anti-GDF-8 antibody would havegreater glucose uptake, increased glycolysis and glycogen synthesis,lower free fatty acids and triglycerides in the serum as compared tomice receiving placebo treatment.

The inhibitory antibodies against GDF-8 are also used to prevent and/orto reduce severity and/or the symptoms of the disease. It is anticipatedthat the anti-GDF-8 antibodies would be administered as a subcutaneousinjection as frequently as once per day and as infrequently as once permonth. Treatment duration could range between one month and severalyears.

To test the clinical efficacy of anti-GDF-8 in humans, subjectssuffering from or at risk for type 2 diabetes are identified andrandomized to a treatment group. Treatment groups include a placebogroup and one to three groups receiving antibody (different doses).Individuals are followed prospectively for one month to three years toassess changes in glucose metabolism. It is anticipated that individualsreceiving treatment would exhibit an improvement.

The antibodies are administered as the sole active compound or incombination with another compound or composition. When administered asthe sole active compound or in combination with another compound orcomposition, the dosage may be between approximately 1 μg/kg and 20mg/kg, depending on the severity of the symptoms and the progression ofthe disease. The appropriate effective dose is selected by a treatingclinician from the following ranges: 1 μg/kg and 20 mg/kg, 1 μg/kg and10 mg/kg, 1 μg/kg and 1 mg/kg, 10 μg/kg and 1 mg/kg, 10 μg/kg and 100μg/kg, 100 μg and 1 mg/kg, and 500 μg/kg and 1 mg/kg. Exemplarytreatment regimens and outcomes are summarized in Table 5.

TABLE 5 Examples of Clinical Cases Status prior to Treatment Patient No.treatment Regimen Outcome Patient 1 No clinical 0.01-1 mg/kg Preventionof signs, family every 4 weeks type 2 diabetes history of type 2 for 48weeks diabetes Patient 2 Mild clinical 0.01-20 mg/kg Improved insulinsigns of weekly for 4 tolerance and syndrome X more weeks glucosemetabolism, and lower blood pressure Patient 3 Advanced stage 0.01-20mg/kg Improvement of of type 2 twice weekly for clinical signs, diabetes6 or more reduction in weeks severity of symptoms and/or increase inmuscle mass/ body fat ratio Patient 4 Severe insulin 0.01-20 mg/kgImprovement of resistance daily for 6 or clinical signs, and/obesitymore weeks reduction in severity of symptoms and/or decrease in body fat

1-54. (canceled)
 55. An isolated nucleic acid molecule encoding anantibody that specifically binds to amino acids 1-50 of the polypeptideof SEQ ID NO: 15, wherein the antibody reduces Growth andDifferentiation Factor-8 (GDF-8) activity.
 56. The nucleic acid moleculeof claim 1, wherein the antibody specifically binds to amino acids 1-25of the polypeptide of SEQ ID NO:
 15. 57. The nucleic acid molecule ofclaim 1, wherein the antibody specifically binds to amino acids 1-15 ofthe polypeptide of SEQ ID NO:
 15. 58. An isolated nucleic acid moleculeencoding an antibody that specifically binds to the polypeptide of SEQID NO: 2, wherein the antibody reduces GDF-8 activity.
 59. An isolatednucleic acid molecule encoding an antibody that specifically binds tothe polypeptide of SEQ ID NO: 3, wherein the antibody reduces GDF-8activity.
 60. An isolated nucleic acid molecule encoding an antibodythat specifically binds to the polypeptide of SEQ ID NO: 5, wherein theantibody reduces GDF-8 activity.
 61. An isolated nucleic acid moleculeencoding an antibody that specifically binds to the polypeptide of SEQID NO: 8, wherein the antibody reduces GDF-8 activity.
 62. An isolatednucleic acid molecule encoding an antibody having the same amino acidsequence as an antibody produced by a cell having ATCC DepositDesignation Number PTA-4236.
 63. An isolated nucleic acid moleculeencoding an antibody that specifically binds to an amino acid sequenceselected from the group consisting of (a) amino acids 1-50, 1-25, or1-15 of SEQ ID NO:15; (b) the polypeptide of SEQ ID NO: 2; (c) thepolypeptide of SEQ ID NO: 3 (d) the polypeptide of SEQ ID NO: 5; and (e)the polypeptide of SEQ ID NO: 8, wherein the nucleic acid encodes aheavy chain variable region that comprises an amino acid sequence thatis at least 85% identical to SEQ ID NO:
 1. 64. The nucleic acid moleculeof claim 63, wherein the nucleic acid molecule comprises the nucleotidesof SEQ ID NO:
 6. 65. An isolated cell comprising the nucleic acid of anyof claims 55-64, wherein the cell produces an antibody that reduces aGDF-8 activity.
 66. A method of increasing trabecular bone density in apatient, said method comprising administering a therapeuticallyeffective amount of an isolated antibody that specifically binds toamino acids 1-50 of the polypeptide of SEQ ID NO: 15, wherein theantibody reduces a GDF-8 activity, thereby increasing trabecular bonedensity.
 67. The method of claim 66, wherein the antibody binds to theGDF-8 protein in the region from amino acid 1 to amino acid 25 of thepolypeptide of SEQ ID NO:
 15. 68. The method of claim 66, wherein theantibody binds to the GDF-8 protein in the region from amino acid 1 toamino acid 15 of the polypeptide of SEQ ID NO:
 15. 69. The method ofclaim 66, wherein the antibody binds to the polypeptides of SEQ ID NO:2.
 70. The method of claim 66, wherein the antibody binds to thepolypeptides of SEQ ID NO:
 3. 71. The method of claim 66, wherein theantibody binds to the polypeptides of SEQ ID NO:
 5. 72. The method ofclaim 66, wherein the antibody binds to the polypeptides of SEQ ID NO:8.
 73. The method of claim 66, wherein the antibody recognizes the GDF-8latent complex, GDF-8 in complex with follisatin, or GDF-8 in complexwith a follistatin related protein.
 74. The method of claim 66, whereinthe antibody is produced by a cell having ATCC Deposit DesignationNumber PTA-4236.
 75. The method of claim 66, wherein the antibody ischimeric.
 76. The method of claim 66, wherein the antibody is humanized.77. The method of of claim 66, wherein the antibody is chosen from asingle-chain, Fab, F(ab′)₂, and Fv.
 78. The method of claim 66, whereinthe antibody is chosen from IgA, IgD, IgE, IgG, and IgM.